Designer protein assemblies with tunable phase diagrams in living cells

Abstract

Protein self-organization is a hallmark of biological systems. Although the physicochemical principles governing protein–protein interactions have long been known, the principles by which such nanoscale interactions generate diverse phenotypes of mesoscale assemblies, including phase-separated compartments, remain challenging to characterize. To illuminate such principles, we create a system of two proteins designed to interact and form mesh-like assemblies. We devise a new strategy to map high-resolution phase diagrams in living cells, which provide self-assembly signatures of this system. The structural modularity of the two protein components allows straightforward modification of their molecular properties, enabling us to characterize how interaction affinity impacts the phase diagram and material state of the assemblies in vivo. The phase diagrams and their dependence on interaction affinity were captured by theory and simulations, including out-of-equilibrium effects seen in growing cells. Finally, we find that cotranslational protein binding suffices to recruit a messenger RNA to the designed micron-scale structures.

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Fig. 1: A synthetic system for controlled phase separation in living cells.
Fig. 2: Characterization of phase diagrams in living cells.
Fig. 3: Influence of affinity on phase separation in vivo.
Fig. 4: Cotranslational binding of a nascent chain directs mRNA localization.

Data availability

We provide single-cell measurements of YFP and RFP concentrations for all phase diagrams in two Supplementary Excel tables. Other data are available from the authors upon request. Source Data are provided with this paper.

Code availability

Code and custom scripts used in this work are available from the authors upon request. We used the open source package oxDNA (v.2.4) to run the sedimentation simulations.

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Acknowledgements

We thank J. Gerst and R. R. Nair (Weizmann Institute) for sharing plasmids of the MS2 system, S. Schwartz (Weizmann Institute) for the bRA89 plasmid, F. Sciortino and H. Hofmann for helpful discussions and suggestions and H. Greenblatt for help with computer systems. E.D.L. acknowledges support by the Israel Science Foundation (no. 1452/18), by the European Research Council under the European Union’s Horizon 2020 research and innovation program (grant agreement no. 819318), by the HFSP Career Development Award (award no. CDA00077/2015), by a research grant from A.-M. Boucher and by research grants from the Estelle Funk Foundation, the Estate of Fannie Sherr, the Estate of Albert Delighter, the Merle S. Cahn Foundation, Mrs. Mildred S. Gosden, the Estate of Elizabeth Wachsman and the Arnold Bortman Family Foundation. E.D.L. is an incumbent of the Recanati Career Development Chair of Cancer Research. L.R. acknowledges support from the European Commission (Marie Skłodowska-Curie Fellowship, no. 702298-DELTAS). S.A.S. thanks the BSF and the ISF program and acknowledges the historical generosity of the Perlmann family foundation. S.K.N. acknowledges support from the Koshland foundation and Department of Atomic Energy (DAE), India. E.L. and L.R. thank the Vienna Scientific Cluster (VSC) for computing time.

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Authors

Contributions

M.H., J.M.G. and E.D.L. designed the research and synthetic protein system. M.H. and J.M.G. performed the experiments with help from Y.N. E.L., L.R. and J.K.P.D developed the theoretical framework for modeling the system based on patchy particles. S.K.N. and S.A.S. developed the theoretical framework for modeling the system based on a lattice model. A.S. wrote the image analysis scripts. E.S. carried out electron microscopy experiments. M.H. and E.D.L. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Lorenzo Rovigatti or Samuel A. Safran or Jonathan P. K. Doye or Emmanuel D. Levy.

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The authors declare no competing interests.

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Extended data

Extended Data Fig. 1 The components do not form condensates when expressed individually.

Haploid cells expressing only one of the building blocks show a homogenous distribution of fluorescence throughout the cytoplasm. The left-most image shows cells expressing the dimer component lacking the Im2 domain. The next images show cells expressing the variants of the dimer component in the absence of the tetramer component. The right-most image shows cells expressing the tetramer component in the absence of the dimer component. This result was replicated three times.

Extended Data Fig. 2 The synthetic condensates are not membrane-bound.

a, Transmission electron microscopy (TEM) micrograph of fixed and sectioned yeast shows a condensate formed by our minimal system, in the cytoplasm. b, The yellow arrow points to one of several 10 nm gold-labeled anti-GFP antibodies, confirming the identity of the designed compartments. White arrows highlight the lack of membrane surrounding the compartment. c, Scanning electron microscopy micrograph of cells frozen at high-pressure and cryo-fractured reveals the mosaic of amorphic cytoplasm. The region outlined by white carets exhibits a distinct ultrastructure d, Increased magnification of a suspected condensate within the cytoplasm, outlined with white carets. This ultrastructure has no visible membrane. Scale bar 1 µm. We did not carry independent biological replicates of these electron microscopy experiments.

Extended Data Fig. 3 Impact of affinity on the phase diagram of the dimer-tetramer system.

a, We used a lattice model (Supplementary Note, Section 1) of the dimer-tetramer system. In the square lattice, concentration is measured by fractional occupancy of edges and vertices by dimers and tetramers respectively. We calculated the binodal of this system in the plane corresponding to the fractional occupancy of dimer (x-axis) and tetramer (y-axis). Affinity increases in panels from left to right, where μ is the binding energy in units of kT of a linker and one arm of the tetravalent molecule. Higher affinity (larger μ) increases the fraction of the phase-separated region. b, We used mean-field theoretical calculations of patchy particles matching the geometry of the proteins. The binodal is calculated in the plane corresponding to the concentration of dimers (x-axis) and tetramers (y-axis). Affinity (which is linked to the energy and entropy associated with the formation of a bond, see Supplementary Note, Section 1) increases from left to right.

Extended Data Fig. 4 Simulations recapitulate the kinetic trapping effect observed experimentally.

a, Sedimentation molecular dynamics simulation of patchy particles. Several simulations were conducted at equilibrium or out-of-equilibrium while sampling different concentrations of dimer and tetramer. The protein osmotic pressure as a function of density was inferred from each simulation and used to evaluate the phase boundaries. b, The phase diagram of the patchy mixture computed with equilibrium and non-equilibrium simulations (squares and circles, respectively).

Extended Data Fig. 5 In vivo phase diagrams and fluorescence recovery profiles observed with different affinities.

a, In vivo phase diagrams observed for five affinities investigated initially. Concentrations correspond to those of the binding sites (not of the dimer and tetramer complexes). The red line highlights the diagonal, where the concentrations of binding sites of dimer and tetramers are equal. The grey dotted lines show the lower limit of concentrations that can be reliably estimated. b, Fluorescence recovery profiles of photobleached condensates for different interaction affinities between the components. Grey lines show individual experiments, the red line corresponds to the mean recovery and the transparent red area indicates the standard error. The mean recovery after 25 seconds and associated standard error are given for each affinity. Source data

Extended Data Fig. 6 Replicating the measurement of in vivo phase diagrams with four additional affinities.

Phase diagrams measured for nine affinities. Five affinities come from replicating experiments shown in Extended Data Fig. 5, and four are new. Concentrations correspond to those of the binding sites (not of the dimer and tetramer complexes). The red line highlights the diagonal, where the concentrations of binding sites of dimer and tetramers are equal. The grey dotted lines show the lower limit of concentrations that can be reliably estimated. Affinities and mutations are indicated above. The N34V, R38T, double N34V/R38T and triple D33L/N34V/R38T mutants were added later to further investigate the out-of-equilibrium effect. The same number of randomly selected cells were plotted in all panels (n=4000) to allow comparing the density of points across plots. Source data

Extended Data Fig. 7 The mRNA coding for the dimer is released from condensates within minutes after the addition of puromycin.

Cells were treated with a final concentration of 10 mM puromycin and mRNA release from the condensate was followed by time-lapse microscopy.

Supplementary information

Supplementary Information

Supplementary Tables 1–3, Figs. 1–5 and Note.

Reporting Summary

Supplementary Video 1

Synthetic condensates expressed in yeast cells. New synthetic condensates appear in budding daughter cells and their size grows over time. The video is representative of at least three independent experiments.

Supplementary Video 2

FRAP on condensates. Condensates involving high- (left) and low- (right) affinity binding domains show slower (left) and faster (right) recovery after photobleaching. The video is representative of at least 13 independent experiments.

Supplementary Video 3

Localization of dimer mRNA in condensates. mRNAs coding for the dimer building block localize in condensates in yeast cells. The video is representative of at least three independent experiments.

Supplementary Video 4

Localization of GB1 mRNA. mRNAs coding for GB1, a protein that does not bind condensates, colocalize with condensates. The video is representative of at least three independent experiments.

Supplementary Video 5

C-terminal variant of the binding domain. mRNAs do not localize at condensates when the binding domain is encoded at the C terminus of the dimer. The video is representative of at least three independent experiments.

Supplementary Video 6

Puromycin. mRNAs detach from condensates in yeast cells treated with puromycin. The video is representative of at least three independent experiments.

Supplementary Video 7

Puromycin + cyclohexamide. mRNAs remain localized at condensates in yeast cells treated with puromycin and cycloheximide. The video is representative of at least three independent experiments.

Source data

Source Data Figs. 2g and 3a and Extended Data Fig. 5

Data for the phase diagrams.

Source Data Extended Data Fig. 6

Data for the phase diagrams.

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Heidenreich, M., Georgeson, J.M., Locatelli, E. et al. Designer protein assemblies with tunable phase diagrams in living cells. Nat Chem Biol 16, 939–945 (2020). https://doi.org/10.1038/s41589-020-0576-z

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